Measuring chip for surface plasmon resonance biosensor and method for producing the same

ABSTRACT

An objective of the present invention is to provide a measuring chip for a surface plasmon resonance sensor that can detect a small amount of target substances in high sensitivity. The present invention provides a measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a biologically active substance immobilized on the surface of said plasma polymerization layer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a surface plasmon resonance biosensor,specifically, a measuring chip for the same and a method for producingthe measurement chip.

2. Background Art

A number of methods using immunological reactions are used in clinicaltests for detecting target substances. Conventional methods are known tobe intricate and require labeling substances. Thus, immunologicalsensors using a surface plasmon resonance biosensor (SPR) is being used,in which no labeling substance is required and a ligand can be detectedwith high sensitivity. This surface plasmon resonance biosensor is basedon the phenomenon that the intensity of a monochromatic light reflectedfrom the interface between an optically transparent substance such asglass and a metal thin-film layer is dependent on the refractive indexof a sample placed on the reflecting side of the metal. Accordingly, asample can be analyzed by measuring the intensity of the reflectedmonochromatic light.

An optical part of a measuring cell for this surface plasmon resonance(surface plasmon resonance biosensor) has a structure shown in FIG. 2.Namely, porous material 5 is formed on metal thin-film 2 formed on glasssubstrate 1, and physiologically active substance 4, such as an enzymeor antibody, is retained or immobilized on the surface or inside ofporous material 5. Examples of porous material 5 to be used includeweaved, knitted or non-woven cloths made of synthetic fibers, naturalfibers, inorganic fibers or the like, and porous inorganic or organicmaterials (see Japanese Patent Laid-open No. 164195/1991). Furthermore,carboxymethyl dextran is used as a porous material in a commercialproduct (BIAcore 2000, Pharmacia Biosensor).

However, physiologically active substance 4 just exists on the surfaceof porous material 5 and interacts with target substances.

LB (Langmuir-Blodgett) method is occasionally used to immobilizephysiologically active substance 4 on metal thin-film 2 (see JapanesePatent Laid-open No. 288672/1993). However, this method has adisadvantage in that LB membrane binds poorly to a metal thin-film andpeels off together with the physiologically active substance.

Furthermore, Japanese Patent Laid-open No. 264843/1997 disclosesmeasuring cells for a surface plasmon resonance biosensor.

SUMMARY OF THE INVENTION

The present inventors have now found that sensitivity of a measuringchip for a surface plasmon resonance sensor is extremely improved whenonly a small amount of a physiologically active substance is immobilizedon a specific plasma polymerization layer.

An objective of the present invention is to provide a measuring chip fora surface plasmon resonance sensor that can detect a small amount oftarget substances in high sensitivity.

Another objective of the present invention is to provide a measuringcell for a surface plasmon resonance sensor that can detect a smallamount of target substances in high sensitivity.

Further objective of the present invention is to provide a method forproducing said measuring chip.

The present invention provides a measuring chip for a surface plasmonresonance sensor comprising a metal layer and one or more plasmapolymerization layers formed on said metal layer.

The present invention also provides a measuring chip for a surfaceplasmon resonance sensor comprising a metal layer, one or more plasmapolymerization layers formed on said metal layer, and a biologicallyactive substance immobilized on the surface of said plasmapolymerization layer.

The present invention also provides a measuring cell for a surfaceplasmon resonance sensor comprising said measuring chip.

The present invention also provides a method for producing a measuringchip for a surface plasmon resonance sensor comprising the steps offorming a metal layer on an optically transparent substrate, forming oneor more plasma polymerization layers on said metal layer, and thenimmobilizing a biologically active substance on the surface of saidplasma polymerization layer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic sectional view of one embodiment of the measuringchip for a surface plasmon resonance sensor according to the presentinvention.

FIG. 2 is a schematic sectional view of an optical part of a measuringchip for a conventional surface plasmon resonance biosensor. 1:Transparent substrate; 2: Metal thin-film; 3: Plasma polymerizationlayer; 4: Physiologically active substance; 5: Porous material.

FIGS. 3 (a) and (b) each show a schematic sectional view of an opticalpart of a measuring chip for a surface plasmon resonance biosensor. (a)shows immobilization of an Fab fragment of an antibody. (b) showsimmobilization of an F(ab′)₂ fragment of an antibody. 1: Transparentsubstrate; 2: Metal thin-film; 3: Plasma polymerization layer; 4:Physiologically active substance.

FIG. 4 is a schematic sectional view of an optical part of a measuringchip for a surface plasmon resonance biosensor.

FIG. 5 illustrates a surface plasmon resonance biosensor. 7: Cartridgeblock; 8: Light source; 9: Detector; 10: Measuring chip; 71: Measuringcell; 72, 73: Flow routes; 80: Incident light; 90: Reflecting light.

FIG. 6 illustrates a reflected light intensity curve before and afterplasma polymerization membrane formation.

FIG. 7 illustrates a schematic view showing the apparatus used inExample 1.

FIG. 8 shows the relationship between the concentration of thecomplementary DNA and RU in Example 1.

FIG. 9 shows the relationship between the concentration of thecomplementary DNA and RU in Example 2.

FIG. 10 shows the relationship between the concentration of thecomplementary DNA and RU in Example 3.

FIG. 11 shows the relationship between the concentration of thecomplementary DNA and RU in Example 4.

FIG. 12 shows the relationship between the concentration of the HSAantigen and RU in Example 5.

FIG. 13 shows the relationship between the concentration of the BSAantigen and RU in Example 6.

FIG. 14 shows the relationship between the concentration of the sugarand RU in Example 7.

FIG. 15 shows the relationship between the concentration of the BSAantigen and RU in Example 8.

FIG. 16 shows the relationship between the concentration of the BSAantigen and RU in Example 9.

FIG. 17 shows the relationship between the concentration of the BSAantigen and RU in Example 10.

FIG. 18 shows the relationship between the concentration of the BSAantigen and RU in Example 11.

FIG. 19 shows the relationship between the concentration of the HSAantigen and RU in Example 12.

FIG. 20 shows the relationship between the concentration of the HSAantigen and RU in Example 13.

FIG. 21 shows the relationship between the concentration of the HSAantigen and RU in Example 14.

FIG. 22 shows the relationship between the concentration of the HSAantigen and RU in Example 15.

FIG. 23 shows the relationship between the concentration of the HSAantigen and RU in Example 16.

FIG. 24 shows the relationship between the concentration of thecomplementary DNA and RU in Example 17.

FIG. 25 shows the relationship between the concentration of the HSAantigen and RU in Example 18.

FIG. 26 shows the relationship between the concentration of skatole andRU in Example 19.

FIG. 27 shows the relationship between the concentration of the HSAantigen and RU in Example 20.

FIG. 28 shows the relationship between the concentration of the HSAantigen and RU in Example 21.

DETAILED DESCRIPTION OF THE INVENTION

The measuring chip for a surface plasmon resonance sensor (“measuringchip”) may have optically transparent substrate (transparent substrate)1, metal thin-film 2 formed on transparent substrate 1, plasmapolymerization layer 3 formed on metal thin-film 2, and physiologicallyactive substance 4 immobilized on the surface of plasma polymerizationlayer 3 as shown in FIG. 1.

Transparent substrate 1 can be any substrate customarily used in ameasuring chip for a surface plasmon resonance sensor. Generally,substrates made of materials that are transparent to a laser beam suchas glass can be used. The thickness of the substrate can be about 0.1 to5 mm.

Metal thin-film 2 is not particularly restricted, provided it can inducesurface plasmon resonance. Examples of the metal to be used for metalthin-film 2 include gold, silver and platinum. They can be used alone orin combination. Furthermore, for better adhesion to transparentsubstrate 1, an auxiliary layer made of chrome or the like may be setbetween transparent substrate 1 and the layer made of gold, silver orthe like.

The thickness of metal thin-film 2 is preferably 100 to 2000 angstroms,most preferably 100 to 500 angstroms. If the thickness exceeds 3,000angstroms, surface plasmon phenomena of the medium cannot besufficiently detected. Furthermore, when an auxiliary layer made ofchrome or the like is formed, the thickness of the auxiliary layer ispreferably 30 to 50 angstroms.

Plasma polymerization layer 3 can be formed by plasma polymerization ofa monomer material for three-dimensional cross-linking. A monomermaterial to be used in the present invention can be any material thatcan immobilize a physiologically active substance by plasmapolymerization.

Examples of a monomer material for a plasma polymerization layer includecompounds of formula (I):CH₃—(CH₂)_(n)—NH₂ (wherein n is an integer from 1 to 6)  (I)and compounds of formula (II):NH₂—(CH₂)_(n)—NH₂ (wherein n is an integer from 1 to 6)   (II)and compounds which comprise carbon (C), hydrogen (H) and nitrogen (N)and have double bonds or triple bonds, such as acetonitrile, vinylamineand pyridine.

Furthermore, when a cross-linking reagent or a condensation reagent isused as a linking layer, a compound further containing sulfur (S),oxygen (O) or silicon (Si) can be used as a monomer material. Generally,a compound appropriately containing any two or more elements selectedfrom carbon (C), hydrogen (H), nitrogen (N), sulfur (S), oxygen (O) andsilicon (Si) can be used. In addition, a halogen gas or a rare gas canbe used as a monomer material.

In the present invention, a compound containing nitrogen can be used asa monomer material. Examples of the compound containing nitrogen includenitrogen N₂; ammonium; hydrazine; pyridine; compounds of formulae (I)and (II) such as ethylenediamine NH₂(CH₂)₂NH₂, hexamethylenediamine NH₂(CH₂)₆NH₂, n-propylamine CH₃(CH₂)₂NH₂ and monoethylamine CH₃(CH₂)NH;compounds of formula (CH₃)₃(CH₂)_(n)N n=0 to 17) such as triethylamine(C₂H₅)₃N; compounds of formula (CH₃)₂(CH₂)_(n)NH (n=0 to 17) such asdiethylamine (C₂H₅)₂NH; compounds of formula CH₂═CH(CH₂)_(n)NH₂ (n=0 to17) such as allylamine CH₂═CHCH₂NH₂; compounds of formula CH₃(CH₂)_(n)CN(n=0 to 17) such as acetonitrile CH₃CN; compounds of formulaCH₃(CH₂)_(n)CN; propargylamine CHCCH₂NH₂; compounds of formula CHC(CH₂)_(n)NH₂; acrylamide; aniline; acrylonitrile; 1,2,4-triazole; and5-amino-1H-tetrazole.

Further examples of of the compound containing nitrogen include thefollowing:

RaNRb₂:

Ra is H or CH₃(CH₂)_(n) (n=0 to 17),

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group, and

Rb is H or CH₃(CH₂)_(n) (n=0 to 17),

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group;

RaNRc:

Rc is H or CH₃(CH₂)_(n)CH (n=0 to 17), or CH₂,

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group;

RdN:

Rd is CH₃(CH₂)_(n)C (n=0 to 17) or CH,

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group;

ReNRfNRg₂:

Re is H or CH₃(CH₂)_(n) (n=0 to 17),

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group,

Rf is (CH₂)_(n) (n=0 to 17),

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group,

Rg is H or CH₃(CH₂)_(n) (n=0 to 17),

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group;

RhNRiNRj

Rh is H or CH₃(CH₂)_(n) (n=0 to 17) or CH₃(CH₂)_(n) CH (n=0 to 17) orCH)₂,

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group,

Ri is (CH₂)_(n) (n=0 to 17) or CH (CH₂)_(n) CH (n=0 to 17),

and includes a group having a double bond a triple bond or both in thechain, and further a branched or cyclized group,

Rj is H or CH₃(CH₂)_(n) CH (n=0 to 17) or CH₃(CH₂)_(n)CH (n=0 to 17) orCH₂ or CH₃(CH₂)_(n) C (n=0 to 17) or CH,

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group;

NRkN:

Rk is C(CH₂)_(n)C (n=0 to 17),

and includes a group having a double bond or a triple bond or both inthe chain, and further a branched or cyclized group.

In the present invention, a compound containing sulfur can be used as amonomer material. Examples of the compound containing sulfur includehydrogen sulfide; carbon disulfide thiophene; compounds of formulaCH₃S(CH₂)_(n)CH₃ (n=0 to 17) such as dimethyl sulfide (CH₃)₂S; compoundsof formula CH₃(CH₂)_(n)SS(CH₂)_(m)CH₃ (n=0 to 17, m=0 to 17) such asmethyl disulfide CH₃SSCH₃; compounds of formula CH₃(CH₂)_(n)SH (n=0 to17) such as ethanethiol CH₃CH₂SH; compounds of formula SH(CH₂)_(n)SH(n=1 to 17) such as ethanedithiol SH(CH₂)₂SH; mercaptoethanol; anddithreitol.

Furthermore, compounds having one of or two or more of groups including—COOH, —CHO, —SH, —NH₂, —OH, ═NH, CONH₂, —NCO, —CH═CH₂, ═C═O and

can be used as a monomer material. Examples of such compounds includecysteine, glutathione, formyl succinate, aminobenzoate, aminohexanate,mercaptobenzoate, and compounds having —C≡CCH₂OH.

In the present invention, a compound containing a halogen can 10 be usedas a monomer material. Examples of the compound containing a halogen fora plasma polymerization layer include tetrafluoroethylene,chlorobenzene, hexachlorobenzene, hexafluorobenzene, and vinyl fluoride.

In the present invention, an organic metal compound can be used as amonomer material. Examples of an organic metal compound for the plasmapolymerization layer include an organic silicon compounds such astetramethylsilane, tetramethyldisiloxane, hexamethyldisiloxane,hexamethyldisilazane, hexamethylcyclotrisilazane,dimethylaminotrimethylsilane, trimethylvinylsilane, tetramethoxysilane,aminopropyltriethoxysilane, octadecyldiethoxymethylsilane,hexamethyldisilane and divinyltetramethyldisiloxane.

Compounds of formulae (I) and (II) having no double bond or triple bondare preferably used because a layer is formed slowly so that theresulting layer is more homogeneous, compared with compounds havingdouble bonds or triple bonds.

The thickness of plasma polymerization layer 3 is preferably 100 to 3000angstroms, most preferably 500 to 1000 angstroms.

Plasma polymerization layer 3 can be formed by plasma treatment to aresulting plasma layer with a polymeric or non-polymeric monomer.Examples of such non-polymeric monomer material include nitrogen,ammonium, hydrazine, hydrogensulfide, hydrogendisulfide, oxygen,hydrogen, water, halogen gas, and rare gas (e.g., argon, neon, helium,krypton, and xenon).

Furthermore, a mixture of various kinds of monomer materials can be usedas a monomer material. Plasma polymerization layer 3 can also be formedby lamination techniques and optionally using a mixture as a monomermaterial.

Plasma polymerization layer 3 of the present invention has the followingadvantages:

1) The layer is pinhole-free, amorphous, and dense.

2) A thin homogeneous layer down to about 500 angstroms can be made,which exhibits extremely little fluctuation in its refractive index.

3) By changing the kind of plasma gas, not only a change in thethickness of the layer but also surface modification and surfaceimprovement, such as introduction of functional groups, and control ofthe density of the functional groups to be introduced can be attained.

4) The layer can be formed in combination with semiconductor techniquessince it is formed under dry conditions.

5) The layer has excellent drug tolerance, heat tolerance, mechanicalproperties, and stability.

Furthermore, in the case of a sensor chip for SPR, in which a metalthin-film is essential, the metal thin-film and the plasmapolymerization layer can be formed in the same chamber. Thus, themanufacturing process can be simplified.

It is also advantageous to attain surface improvement, such asintroduction of a functional group, by further exposing a resultingplasma polymerization layer to plasma treatment with a non-polymeric orpolymeric monomer. The plasma polymerization treatment is intended toinclude a treatment with not only a non-polymeric monomer and aninactive monomer but also a polymeric monomer.

Physiologically active substance 4 to be immobilized is not particularlylimited, provided it reacts interacts with a target substance to bemeasured. Examples of physiologically active substance 4 include nucleicacids (e.g., DNA, RNA, and PNA); non-immune proteins (e.g., avidin(streptoavidin), biotin or a receptor); immunoglobulin-binding proteins(e.g., protein A, protein G and a rheumatoid factor (RF)); sugar-bindingproteins (e.g., lectin); sugar-recognizing sugar chains; fatty acids orfatty acid esters (e.g., stearic acid, alachidic acid, behenic acid,ethyl stearate, ethyl arachidate, and ethyl behanate); polypeptides oroligopeptides having ligand binding activity; immune proteins (e.g., anantibody); and enzyme.

When an antibody is used as physiologically active substance 4, Fcfragments of the antibody can be immobilized only on the surface ofplasma polymerization layer 3 and the antibody is formed in amonomolecular layer as shown in FIG. 1. However, since the sensitivityand the reaction rate decrease as Fab fragments of the antibody areseparated from plasma polymerization layer 3, Fab fragments (FIG. 3 (a))or F(ab′)₂ fragments (FIG. 3 (b)) can be immobilized directly on plasmapolymerization layer 3 as shown in FIG. 3 to improve the sensitivity andthe reaction rate.

The thickness of physiologically active substance 4 depends on the sizeof the physiologically active substance itself, but is preferably 100 to3000 angstroms, most preferably 100 to 1000 angstroms.

In the present invention, the physiologically active substance can beimmobilized on the plasma polymerization layer through linking agents.

FIG. 4 is a schematic illustration showing one embodiment of themeasuring chip according to the present invention. The measuring chiphas covalent bond layer 6 between plasma polymerization layer 3 andphysiologically active substance 4. Substance 4 is immobilized on plasmapolymerization layer 3 via covalent layer 6. The covalent bond can beformed with a cross-linking reagent or a condensation reagent.

The cross-linking reagent or a condensation reagent is not particularlyrestricted, provided it can covalently and firmly immobilize substance4. They can be used alone or in combination.

Examples of such cross-linking reagents include glutaraldehyde, periodicacid, N-succinimidyl-2-maleimidoacetic acid,N-succinimidyl-4-maleimidobutyric acid,N-succinimidyl-6-maleimidohexanic acid,N-succinimidyl-4-maleimidomethylcyclohexan-1-carboxylic acid,N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid,N-succinimidyl-4-maleimidomethylbanzoic acid,N-succinimidyl-3-maleimidobenzoic acid,N-sulfosuccinimidyl-3-maleimidobenzoic acid,N-succinimidyl-4-maleimidophenyl-4-butyric acid,N-sulfosuccinimidyl-4-maleimidophenyl-4-butyric acid,N,N′-oxydimethylene-dimaleimide, N,N′-o-phenylene-dimaleimide,N,N′-m-phenylene-dimaleimide, N,N′-p-phenylene-dimaleimide,N,N′-hexamethylene-dimaleimide, N-succinimidylmaleimidocarboxylic acid,N-succinimidyl-S-acetylmercaptoacetic acid,N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinicanhydride, methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido,bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acidstilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon,1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,p-azidophenacylbromide, p-azidophenylglyoxal,N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate,N-(4-maleimidobutyloxy)sulfosuccinimide sodium salt,N-(6-maleimidocaproyloxy)sulfosuccinimide sodium salt,N-(8-maleimidocaproyloxy)sulfosuccinimide sodium salt,N-(11-maleimidoundecanoyloxy)sulfosuccinimide sodium salt,N-[2-(1-piperazinyl)ethyl]maleimide bichloric acid, bisdiazobenzidine,hexamethylenediisocyanate, toluenediisocyanate,hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimide,N,N′-polymethylenebisiodoacetamide, 2,4-dinitrobenzenesulfonate sodiumsalt, and diazo compounds. Glutaraldehyde is preferable as across-linking reagent.

Examples of such condensation reagents include carbodiimide derivativesrepresented by formula RN═C═NR (or R′), N-hydroxysuccinimide,tri-n-butylamine, butyl chloroformate, and isobutyl isocyanide.

By introducing covalent layer 6 to the measuring cell to firmly mobilizephysiologically active substance 4 via covalent bonds, substance 4 canbe maintained immobilized when the measuring cell is washed, whichenables the cell to be used for repetitive measurements for anotheradvantageous feature. The thickness of covalent layer 6 is preferably 10to 100 angstroms, most preferably 10 to 20 angstroms.

The physiologically active substance can also be immobilized byhydrophobic bond, by integrating substance 4 into a plasmapolymerization layer or by an additional plasma treatment.

A preferred group of the measuring chip according to the presentinvention is a measuring chip comprising a metal layer, one or moreplasma polymerization layers formed on said metal layer, and an immuneprotein or enzyme immobilized on the surface of said plasmapolymerization layer, wherein said plasma polymerization layer comprisesa monomer material selected from the group consisting of pyridine,triethylamine, diethylamine, allylamine, acrylamide, aniline,acrylonitrile, 1,2,4-triazole, 5-amino-1H-tetrazole, and acetonitrile,

Another preferred group of the measuring chip according to the presentinvention is a measuring chip comprising a metal layer, one or moreplasma polymerization layers formed on said metal layer, and an immuneprotein or enzyme immobilized on the surface of said plasmapolymerization layer, wherein said plasma polymerization layer comprisesa monomer material selected from the group consisting of pyridine,triethylamine, diethylamine, allylamine, acrylamide, aniline,acrylonitrile, 1,2,4-triazole, 5-amino-1H-tetrazole, and acetonitrileand wherein said immune protein or enzyme is immobilized on said plasmapolymerization layer through a cross-linking reagent or a water-solublecondensation reagent.

A cross-linking reagent for the preferred group above can be selectedfrom the group consisting of glutaraldehyde,N-succinimidyl-4-maleimidomethylbanzoic acid,N-succinimidyl-3-maleimidobenzoic acid,N-succinimidyl-4-maleimidophenyl-4-butyric acid,N,N′-oxydimethylene-dimaleimide, N,N′-m-phenylene-dimaleimide,N,N′-p-phenylene-dimaleimide, N,N′-hexamethylene-dimaleimide,N-succinimidylmaleimidocarboxylic acid,N-succinimidyl-S-acetylmercaptoacetic acid,N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinicanhydride, methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido,bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acidstilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon,1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,p-azidophenacylbromide, p-azidophenylglyoxal,N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate,bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate,hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimido,N,N′-polymethylenebisiodoacetoamide, and diazo compounds; or saidcondensation reagent is one or more compounds selected from the groupconsisting of carbodiimide derivatives represented by RN═C═NR (or R′),N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, andisobutyl isocyanide.

The measuring chip according to the present invention can be formed asfollows:

First, metal thin-film 2 is formed on transparent substrate 1. Metalthin-film 2 can be formed by conventional methods such as sputtering,CVD, PVD, or vacuum evaporation.

Second, plasma polymerization layer 3 is formed on metal thin-film 2.Plasma polymerization layer 3 can be formed by plasma polymerizationusing a plasma polymerization apparatus. The rate of plasma formation ispreferably 100 to 3000 angstroms/min, most preferably 500 to 1000angstroms/min. If the rate exceeds 3000 angstroms/min, it becomesdifficult to obtain a smooth plasma polymerization layer. Morespecifically, the plasma 35 polymerization can be preferably carried outat a monomer material flow rate of 0.05 to 100 sccm at a roomtemperature or at a temperature of 10 to 20° C. at a pressure between1.0×10⁻² and 1.0×10² Pa using a discharge power of 20 to 300 W at adischarge frequency of 10 MHz or 13.56 MHz. However, polymerizationconditions are not restricted to the conditions above.

After formation of plasma polymerization layer 3, physiologically activesubstance 4 is finally immobilized on plasma polymerization layer 3.Immobilization can be done by conventional methods. For example, aspecified amount of physiologically active substance 4 can beimmobilized by contacting it with plasma polymerization layer 3 for aspecified period of time. If the measuring cell is a flow-cell type, aspecified volume of the physiologically active substance 4 can beimmobilized by contacting it with plasma polymerization layer 3 bypouring a specified volume for a specified period of time.

When an antibody is used as a physiologically active substance and itsFab fragment is immobilized directly on plasma polymerization layer 3,the same treatment can be done after the antibody is partly digestedwith papain. On the other hand, when the F(ab′)₂ fragment is immobilizeddirectly on plasma polymerization layer 3, the same treatment can bedone after the antibody is partly digested with pepsin.

When covalent bond layer 6 is formed, a cross-linking reagent or acondensation reagent is allowed to be in contact with plasmapolymerization layer 3 in the same manner as with active substance 4,after which substance 4 can be immobilized.

The measuring cell for a surface plasmon resonance sensor according tothe present invention comprises the measuring chip. The measuring chipcan be mounted on an optical part to be optically analyzed. The term“optical part” as used herein refers to a part where a light isprojected and an evanescent wave and a surface plasmon can be induced.

The surface plasmon resonance biosensor according to the presentinvention comprises the measuring cell.

FIG. 5 is a schematic view of one embodiment of the surface plasmonresonance biosensor according to the present invention. The surfaceplasmon resonance biosensor has cartridge block 7, light source 8, anddetector 9 and measuring chip 10 is mounted on cartridge block 7. Theupper side of cartridge block 7 has a hollow and this hollow andmeasuring chip 10 construct measuring cell 71.

The body of measuring chip 10 comprises a transparent substrate, and alayer comprising a metal thin-film, a plasma polymerization layer formedunder said metal film. A physiologically active substance is immobilizedon the surface of said plasma polymerization layer facing the hollow ofcartridge block 7. Measuring cell 71 is constructed from the hollow ofcartridge block 7 and measuring chip 10; and cartridge block 7 has flowroutes 72 and 73 providing passages to the outside of measuring cell 71and cartridge block 7, which makes measuring cell 71 a flow-cell type.However, the present invention is not restricted to this type and abatch type cell can also be used. Using measuring cell 71 of thisflow-cell type, a sample can be measured either continuously orintermittently. In this sensor, the sample flows into measuring cell 71via flow route 72 and is discharged after measurement via flow route 73.The flow rate of the sample is preferably 0. 5 to 5 μl/min. The flowrate is controlled, for example, using a computer-operated pump.

Monochromatic light (incident light 80) is irradiated from light source8 toward the optical part of measuring chip 10 and its reflected light90, which is reflected by metal thin-film 2 set on the reverse side ofmeasuring chip 10, reaches detector 9. Detector 9 can detect theintensity of reflected light 90. Light source 8 and detector 9 are notparticularly restricted, and can be any types customarily used for asurface plasmon resonance biosensor. In the sensor according to thepresent invention, the incident light is wedge-shaped and the lightreflected in different directions can be measured simultaneously.However, the present invention is not restricted to this type of sensor.The configuration of this type does not require a mobile part, therebyproducing excellent stability and durability, and enabling real timemeasurement of samples as well.

The configuration as described above yields a reflected light intensitycurve that forms a trough relative to a given angle of incidence (seeFIG. 6). The trough in the reflected light intensity curve is due tosurface plasmon resonance. Namely, when light is totally reflected atthe interface between the transparent substrate and the exterior ofmeasuring chip 10, a surface wave known as an evanescent wave isgenerated at the interface and a surface wave known as a surface plasmonis also generated on the metal thin-film. Resonance occurs when the wavenumber of these two surface waves coincides and a part of light energyis consumed to excite the surface plasmon, resulting in a decrease inthe intensity of the reflected light. The wave number of the surfaceplasmon is affected by the refractive index of the medium proximate tothe surface of the metal thin-film. Therefore, when the refractive indexof the medium changes due to an interaction between the substance to bemeasured and the physiologically active substance, a surface plasmonresonance is induced to change the angle of incidence. Thus, a change inthe concentration of the substance to be measured can be perceived by ashift of the trough in the reflected light intensity curve. The changein the angle of incidence is called a resonance signal and a change of10⁻⁴ degree is expressed as 1 RU. In the surface plasmon resonancebiosensor of this example, highly effective and reliable measurement canbe done if measuring chip 10 is made to be freely attachable anddetachable and disposable. Furthermore, if a covalent bond layer isprovided between the plasma polymerization layer and the physiologicallyactive substance, measuring chip 10 can be used repeatedly by washingthe inside of measuring cell 71, resulting in a decrease in the cost.

The surface plasmon resonance biosensor of the present invention can beused for quantitative or qualitative analysis, identification of atarget substance present in a sample.

EXAMPLE

The present invention is further illustrated by the following Examplesthat are not intended as a limitation of the invention.

Example 1

A measuring chip having layers shown in FIG. 1 on an optical recognitionpart was constructed.

A glass plate with a thickness of 0.15 mm (18 mm×18 mm) was used for atransparent substrate. A chrome layer and then a gold layer weredeposited on this transparent substrate by sputtering. The sputteringwas carried out at 100 W for 40 seconds for the chrome layer and at 100W for 2 minutes and 30 seconds for the gold layer. The resulting chromelayer was 40 angstroms thick and the resulting gold layer was 500angstroms thick.

A plasma polymerization layer was formed on the metal layers. Anapparatus as shown in FIG. 7 was used for plasma polymerization.Ethanedithiol was used as a monomer material for the plasmapolymerization layer to introduce a thiol group. Conditions for plasmapolymerization were as follows:

Flow volume of monomer material: 15 sccm

Temperature: 15° C.

Pressure: 4.7 Pa

Discharge electric power: 20 W

Discharge frequency: 10 MHz, FM modulation

Duration of discharge: 60 seconds.

Under the conditions described above, a thiol group was introduced onthe surface of plasma polymerization layer. The sensor chip with theintroduced thiol group was mounted on the cartridge block of the surfaceplasmon resonance biosensor and maleimidized avidin (see “UltrahighSensitivity Enzyme Immunoassay” by Eiji Ishikawa) was poured through aflow route into the measuring cell at a flow rate of 5 μl/min forimmobilization on the thiol group on the plasma polymerization layer for60 minutes. 50 μl of 10 pM-biotinized DNA were then poured and the probeDNA was immobilized via the avidin for 10 minutes. A DNA (7.5×10⁻⁷ M)having a DNA sequence complementary to this probe DNA was introduced andafter the reaction, a signal of about 500 RU was obtained. Concentrationof Complementary DNA (μM) 0.00075 0.0075 0.075 0.75 7.5 75 RU 10 25 100500 1000 1100

It was confirmed by an XPS analysis that the resulting membrane has amercapto group.

FIG. 6 shows the reflected light intensity curve before and after theformation of the plasma polymerization layer, which show the intensityof reflected light corresponding to the angle of incidence θ) FIG. 6shows that the plasma polymerization layer is formed on the surface ofthe gold layer. The thickness of the plasma polymerization layer can beestimated from Δθ.

Example 2

The same apparatus and method as in Example 1 were used.

Acetonitrile was used as a monomer material for the plasmapolymerization layer. Conditions for plasma polymerization were asfollows:

Flow volume of monomer material: 1.5 sccm+Ar dilution 15 (sccm)

Temperature: room temperature

Pressure: 4.7 Pa

Discharge electric power: 80 W

Discharge frequency: 13.56 MHz

Duration of discharge: 15 seconds.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and avidin (concentration: 20 μg/ml) was also poured at aflow rate of 5 μl/min to immobilize for 60 minutes. 10 μM biotin-labeledprobe RNA were then poured at a flow rate of 1 μl/min to immobilize theprobe RNA for 10 minutes. DNA (7.5×10⁻⁷ M) having a DNA sequencecomplementary to this probe RNA was introduced and after the reaction, asignal of about 500 RU was obtained. Concentration of Complementary DNA(μM) 0.00075 0.0075 0.075 0.75 7.5 75 RU 8 20 80 400 800 880

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 3

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 2.

Under the conditions described above, a plasma polymerization layer wasformed.

The sensor chip was mounted on the cartridge block of the surfaceplasmon resonance biosensor, 5% glutaraldehyde was poured through a flowroute into the measuring cell at a flow rate of 5 μl/min for 10 minutesand streptoavidin (concentration: 20 82 g/ml) was also poured at a flowrate of 5 μl/min to immobilize for 60 minutes. 10 μM biotin-labeledprobe RNA was then poured at a flow rate of 1 μl/min for 10 minutes toimmobilize the probe RNA. DNA (7.5×10⁻⁷ M) having a DNA sequencecomplementary to this probe RNA was introduced and after the reaction, asignal of about 375 RU was obtained. Concentration of Complementary DNA(μM) 0.00075 0.0075 0.075 0.75 7.5 75 RU 7.5 18.75 75 375 750 825

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 4

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 2 except that propargylamine was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 0.4 MN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide was poured through a flowroute into the measuring cell at a flow rate of 5 μl/min for 10 minutesand avidin (concentration: 20 μg/ml) was also poured at a flow rate of 5μl/min to immobilize for 60 minutes. 10 μM biotin-labeled probe RNA wasthen poured at a flow rate of 1 μl/min for 10 minutes to immobilize theprobe RNA. DNA (7.5×10⁻⁷ M) having a DNA sequence complementary to thisprobe RNA was introduced and after the reaction, a signal of about 450RU was obtained. Concentration of Complementary DNA (μM) 0.00075 0.00750.075 0.75 7.5 75 RU 0.9 22.5 90 450 900 990

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 5

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 4.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and protein A (concentration: 400 μg/ml) was also pouredat a flow rate of 5 μl/min to immobilize for 60 minutes. An anti-HSAantibody (concentration: 400 μl/ml) was then poured at a flow rate of 1μl/min for 10 minutes to immobilize the antibody. An HSA antigen (10μg/ml) complementary to this anti-HSA antibody was introduced and afterthe reaction, a signal of about 250 RU was obtained. Concentration ofHSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU 5 12.5 50 250 500 550

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 6

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 4.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and protein G (concentration: 400 μg/ml) minutes. Ananti-BSA antibody (concentration: 400 μl/ml) was then poured at a flowrate of 1 μl/min for 10 minutes to immobilize the antibody. A BSAantigen (10 μg/ml) complementary to this anti-BSA antibody wasintroduced and after the reaction, a signal of about 225 RU wasobtained. Concentration of BSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU4.5 11.25 45 225 450 495

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 7

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 4.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and mannose-binding lectin (concentration: 200 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes.

A sugar (10 μg/ml) complementary to this mannose-binding lectin wasintroduced and after the reaction, a signal of about 200 RU wasobtained.

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 8

Concentration of sugar (μg/ml) 0.01 0.1 1 10 100 1000 RU 4 10 40 200 400440

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 4 except that pyridine was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-BSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. ABSA antigen (10 μg/ml) complementary to this anti-BSA antibody wasintroduced and after the reaction, a signal of about 187.5 RU wasobtained. Concentration of BSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU3.75 9.375 37.5 187.5 375 412.5

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 9

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 8 except that acrylonitrile was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-BSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. ABSA antigen (10 μg/ml) complementary to this anti-BSA antibody wasintroduced and after the reaction, a signal of about 200 RU wasobtained. Concentration of BSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU4 10 40 200 400 440

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 10

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 9 except that ethanethiol was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor and maleimidized anti-BSA antibodywas poured through a flow route at a flow rate of 5 μl/min to immobilizefor 60 minutes. A BSA antigen (10 μg/ml) complementary to this anti-BSAantibody was introduced and after the reaction, a signal of about 200 RUwas obtained. Concentration of BSA antigen (μg/ml) 0.01 0.1 1 10 1001000 RU 4 10 40 200 400 440

It was confirmed by the XPS analysis that the resulting membrane has amercapto group.

Example 11

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 10 except that thiophene was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor and maleimidized anti-BSA antibodywas poured through a flow route at a flow rate of 5 μl/min to immobilizefor 60 minutes. A BSA antigen (10 μg/ml) complementary to this anti-BSAantibody was introduced and after the reaction a signal of about 187.5RU was obtained. Concentration of BSA antigen (μg/ml) 0.01 0.1 1 10 1001000 RU 3.75 9.375 37.5 187.5 375 412.5

It was confirmed by the XPS analysis that the resulting membrane has amercapto group.

Example 12

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 11 except that acetonitrile was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-HSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. HSAantigen (10 μg/ml) complementary to this anti-HSA antibody wasintroduced and after the reaction, a signal of about 250 RU wasobtained. Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU5 10 50 250 500 550

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 13

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 12.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and the Fab fragment of an anti-HSA antibody(concentration: 400 μg/ml) was also poured at a flow rate of 5 μl/min toimmobilize for 60 minutes.

A HSA antigen (10 μg/ml) complementary to this Fab fragment of theanti-HSA antibody was introduced and after the reaction, a signal ofabout 275 RU was obtained. Concentration of HSA antigen (μg/ml) 0.01 0.11 10 100 1000 RU 5.5 11 55 275 550 605

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 14

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 13.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and the F(ab)₂ fragment of an anti-HSA antibody(concentration: 400 μg/ml) was also poured at a flow rate of 5 μl/min toimmobilize for 60 minutes.

A HSA antigen (10 μg/ml) complementary to this F(ab)₂ fragment of theanti-HSA antibody was introduced and after the reaction, a signal ofabout 300 RU was obtained. Concentration of HSA antigen (μg/ml) 0.01 0.11 10 100 1000 RU 6 12 60 300 600 660

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 15

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were as follows:

(1) Monomer: hexadiene Flow volume of monomer material: 1.5 sccm +Ardilution 15 (sccm)

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 80 W

Discharge frequency: 13.56 MHz

Duration of discharge: 15 seconds;

(2) Monomer: ethylenediamine

Flow volume of monomer material: 1.5 sccm

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 80 W

Discharge frequency: 13.56 MHz

Duration of discharge: 5 seconds.

The targeted surface was obtained by the two-step process above.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-HSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. AHSA antigen (10 μg/ml) complementary to this anti-HSA antibody wasintroduced and after the reaction, a signal of about 250 RU wasobtained. Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU5 10 50 250 500 550

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 16

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were as follows:

(1) Monomer: hexamethyldisiloxane

Flow volume of monomer material: 1.5 sccm+Ar dilution 15 (sccm)

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 80 W

Discharge frequency: 13.56 MHz

Duration of discharge: 15 seconds;

(2) Monomer: ethylenediamine

Flow volume of monomer material: 1.5 sccm

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 80 W

Discharge frequency: 13.56 MHz

Duration of discharge: 5 seconds.

The targeted surface was obtained by the two-step process above.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-HSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. AHSA antigen (10 μg/ml) complementary to this anti-HSA antibody wasintroduced and after the reaction, a signal of about 225RU was obtained.Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU 4.5 9 45225 450 495

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 17

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 2 except that propylamine was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and avidin (concentration: 20 μg/ml) was also poured at aflow rate of 5 μl/min to immobilize for 60 minutes. 10 μM biotin-labeledprobe RNA was poured at a flow rate of 1 μl/min to immobilize the probeRNA for 10 minutes. DNA (7.5×10⁻⁷ M) having a DNA sequence complementaryto this probe RNA was introduced and after the reaction, a signal ofabout 400 RU was obtained. Concentration of Complementary DNA (μM)0.00075 0.0075 0.075 0.75 7.5 75 RU 8 20 80 400 800 880

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 18

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were as follows:

(1) Monomer: propargyl alcohol

Flow volume of monomer material: 1.5 sccm

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 20 W

Discharge frequency: 13.56 MHz

Duration of discharge: 15 seconds;

(2) Monomer: oxygen (plasma treatment)

Flow volume of monomer material: 1.5 sccm

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 20 W

Discharge frequency: 13.56 MHz

Duration of discharge: 5 seconds.

The targeted surface was obtained by the two-step process above.

Under the conditions described above a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, a 0.5 M carbodiimide solution waspoured through a flow route into the measuring cell at a flow rate of 5μl/min for 10 minutes and an anti-HSA antibody (concentration: 400μg/ml) was also poured at a flow rate of 5 μl/min to immobilize for 60minutes. A HSA antigen (10 μg/ml) complementary to this anti-HSAantibody was introduced and after the reaction, a signal of about 250 RUwas obtained. Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 1001000 RU 5 10 50 250 500 550

It was confirmed by the XPS analysis that the resulting membrane has acarboxyl group.

Example 19

The same apparatus and method as in Example 1 were used.

Conditions for plasma polymerization layer formation were the same as inExample 2 except that propagylamine was used as a monomer material.

Under the conditions described above, a plasma polymerization layer wasformed. The sensor chip was mounted on the cartridge block of thesurface plasmon resonance biosensor, 0.5 M carbodiimide was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and behenic acid (concentration: 400 μg/ml) was alsopoured at a flow rate of 5 μl/min to immobilize for 60 minutes. Skatole(10 μg/ml) complementary to this behenic acid was introduced and afterthe reaction, a signal of about 225 RU was obtained. Concentration ofskatole (μg/ml) 0.01 0.1 1 10 100 1000 RU 4.5 9 45 225 450 495

It was confirmed by the XPS analysis that the resulting membrane has aprimary amine.

Example 20

Example 20 shows a formation of hydrophobic bond.

A layer comprising chrome and gold was formed on a transparent substrate(glass plate) by sputtering. A plasma polymerization layer in whichtrifluoroethylene was used as a monomer was then formed on the resultingmetal layer under the following conditions

Flow volume: 1.5 sccm

Temperature: room temperature

Pressure: 5 Pa

Discharge electric power: 50 W

Discharge frequency: 13.56 MHz

Duration of discharge: 30 seconds.

The plasma polymerization layer obtained under the conditions describedabove was hydrophobic. An anti-HSA antibody (concentration: 100 μg/ml)was allowed to flow at a flow rate of 5 μl/min for 60 minutes toimmobilize the antibody via hydrophobic bond. HSA at a specifiedconcentration was further reacted with this antibody-immobilized plasmapolymerization layer. The following results were obtained. Concentrationof HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU 3 6 30 150 300 330

Example 21

Example 21 shows an inclusion of an antibody by plasma polymerization.

A layer comprising chrome and gold was formed on a transparent substrate(glass plate) by sputtering. A plasma polymerization layer in whichpropargyl alcohol was used as a monomer was then formed on the resultingmetal layer under the following conditions:

Flow volume: 1.5 sccm

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 20 W

Discharge frequency: 13.56 MHz

Duration of discharge: 15 seconds.

The plasma polymerization layer obtained under the conditions describedabove was highly hydrophilic. An antibody solution (concentration: 100μg/ml) was spread evenly on this propargyl alcohol plasma polymerizationlayer and after drying, plasma treatment was further carried out on thissurface under the following conditions:

Flow volume: 1.5 sccm

Temperature: room temperature

Pressure: 1.6 Pa

Discharge electric power: 20 W

Discharge frequency: 13.56 MHz

Duration of discharge: 8 seconds.

HSA at a specified concentration was reacted with the membrane in whichthe antibody was thus integrated and immobilized by plasma treatment.The following results were obtained, from which a calibration curvecould be drawn. Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 1001000 RU 5 10 50 250 500 550*Flow rate for HSA: 5 μl/min.

1. A measuring chip for a surface plasmon resonance sensor comprising ametal layer and one or more plasma polymerization layers formed on saidmetal layer.
 2. The measuring chip of claim 1, wherein said metal layercomprises gold, platinum or silver.
 3. The measuring chip of claim 1further comprising a nucleic acid immobilized on the surface of at leastone plasma polymerization layers.
 4. The measuring chip for a surfaceplasmon resonance sensor of claim 1 further comprising a non-immuneprotein immobilized on the surface of at least one plasma polymerizationlayer.
 5. The measuring chip for a surface plasmon resonance sensor ofclaim 1 further comprising a non-immune protein which is avidin,streptoavidin, biotin or a receptor.
 6. The measuring chip for a surfaceplasmon resonance sensor of claim 1, further comprising animmunoglobulin-binding protein immobilized on the surface of at leastone plasma polymerization layer.
 7. The measuring chip for a surfaceplasmon resonance sensor of claim 1, further comprising animmunoglobulin-binding protein which is protein A, protein G, or arheumatoid factor.
 8. The measuring chip for a surface plasmon resonancesensor of claim 1, further comprising a sugar-binding proteinimmobilized on the surface of at least one plasma polymerization layer.9. The measuring chip for a surface plasmon resonance sensor of claim 1further comprising a sugar-binding protein which is a lectin.
 10. Themeasuring chip for a surface plasmon resonance sensor of claim 1,comprising a metal layer, one or more plasma polymerization layersformed on said metal layer, and a sugar-recognizing sugar chainimmobilized on the surface of said plasma polymerization layer.
 11. Themeasuring chip for a surface plasmon resonance sensor of claim 1,comprising a metal layer, one or more plasma polymerization layersformed on said metal layer, and a fatty acid or a fatty acid esterimmobilized on the surface of said plasma polymerization layer.
 12. Themeasuring chip for a surface plasmon resonance sensor of claim 1 furthercomprising a fatty acid or fatty acid ester which is stearic acid,alachidic acid, behenic acid, ethyl stearate, ethyl arachidate, or ethylbehanate.
 13. The measuring chip for a surface plasmon resonance sensorof claim 1 further comprising a polypeptide or oligopeptide havingligand-binding activity immobilized on the surface of said at least oneplasma polymerization layer.
 14. The measuring chip for a surfaceplasmon resonance sensor of claim 1, further comprising a polypeptide oroligopeptide which is produced by genetic engineering or chemicalsynthesis.
 15. The measuring chip of claim 1 further comprising anoptically transparent substrate on which said metal layer is formed. 16.A measuring chip according to claim 1, wherein said one or more plasmapolymerization layer(s) comprises a compound having one or moresubstitutents selected from the group consisting of —COOH, —CHO, —SH,—NH₂, —OH, ═NH, —CONH₂, —NCO, —CH═CH₂, ═C═O, and


17. A measuring chip of claim 1, comprising two or more plasmapolymerization layers.
 18. A measuring chip of claim 1, wherein at leastone plasma polymerization layer is formed from a monomer materialcontaining nitrogen.
 19. The measuring chip for a surface plasmonresonance sensor of claim 1, wherein said plasma polymerization layer isformed from a compound containing nitrogen which is CH₃—(CH₂)_(n)—NH₂(wherein n is an integer from 1 to 6) and/or NH₂—(CH₂)_(n)—NH₂ (whereinn is an integer from 1 to 6).
 20. The measuring chip for a surfaceplasmon resonance sensor of claim 1, comprising a plasma polymerizationlayer which is produced from a compound containing nitrogen selectedfrom the group consisting of pyridine, ethylenediamine,hexamethylenediamine, n-propylamine, monoethylamine, triethylamine,diethylamine, allylamine, acrylamide, aniline, acrylonitrile,1,2,4-triazole, 5-amino-1H-tetrazole, propargylamine and acetonitrile.21. The measuring chip of claim 1, wherein at least one plasmapolymerization layer is formed from a monomer material containingsulfur.
 22. The measuring chip according to claim 1, wherein at leastone plasma polymerization layer is formed from a compound containingsulfur selected from the group consisting of dimethyl sulfide, methyldisulfide, ethanethiol, ethanedithiol, thiophen, mercaptoethanol anddithreitol.
 23. The measuring chip according to claim 1, wherein saidplasma polymerization layer is formed from a monomer compound containinga halogen.
 24. The measuring chip according to claim 1, wherein said atleast one plasma polymerization layer is formed from a monomer materialhaving one or more groups selected from the group consisting of —COON,—CHO, —SH, —NH₂, —OH, ═NH, —CONH₂, —NCO, —CH═CH₂, ═C═O and


25. The measuring chip according to claim 1, wherein said monomermaterial of a plasma polymerization layer is a compound having—C═CCH₂OH.
 26. The measuring chip according to claim 1, wherein saidplasma polymerization layer is formed from a monomer material which is acarbohydrate compound comprising C and H.
 27. The measuring chipaccording to claim 1, wherein said plasma polymerization layer is formedfrom a monomer material which is an organic metal compound.
 28. Ameasuring chip according to claim 27, wherein said organic metalcompound is an organic silicon compound.
 29. A measuring chip accordingto claim 28, wherein said organic silicon compound is selected from thegroup consisting of tetramethylsilane, tetramethyldisiloxane,hexamethyldisiloxane, hexamethyldisilazane, hexamethylcyclotrisilazane,dimethylaminotrimethylsilane, trimethylvinylsilane, tetramethoxysilane,aminopropyltriethoxysilane, octadecyldiethoxymethylsilane,hexamethyldisilane, and divinyltetramethyldisiloxane.
 30. the measuringchip according to claim 1, wherein plasma treatment is applied to formsaid plasma polymerization layer with a polymeric or non-polymericmonomer.
 31. A measuring chip according to claim 30 wherein saidnon-polymeric monomer material is selected from the group consisting ofnitrogen, ammonium, hydrazine, hydrogensulfide, hydrogendisulfide,oxygen, hydrogen, water, halogen gas, and rare gas.
 32. The measuringchip according to claim 1, wherein said plasma polymerization layer isformed from a mixture of two or more different types of monomers. 33.The measuring chip for a surface plasmon resonance sensor of claim 1,further comprising an immune protein or enzyme immobilized on thesurface of said plasma polymerization layer, wherein said plasmapolymerization layer comprises a monomer material selected from thegroup consisting of pyridine, triethylamine, diethylamine, allylamine,acrylamide, aniline, acrylonitrile, 1,2,4-triazole,5-amino-1H-tetrazole, and acetonitrile.
 34. The measuring chip for asurface plasmon resonance sensor of claim 1, further comprising animmune protein or enzyme immobilized on the surface of said plasmapolymerization layer, wherein said plasma polymerization layer comprisesa monomer material selected from the group consisting of a compoundcontaining sulfur, an oxygen containing compound, a carbon containingcompound, a compound containing a halogen, an organic metal compound, anorganic silicon compound, and a carbohydrate compound containing C andH.
 35. A measuring chip according to claim 33, wherein said immuneprotein is an antibody.
 36. measuring chip according to claim 33,wherein said immune protein is a Fab fragment of an antibody.
 37. Ameasuring chip according to claim 33, wherein said immune protein is anF(ab)2 fragment of an antibody.
 38. A measuring chip according to claim3, wherein said nucleic acid is immobilized on said plasmapolymerization layer through a cross-linking reagent or a condensationreagent.
 39. A measuring chip according to claim 4, wherein saidnon-immune protein is immobilized on said plasma polymerization layerthrough a cross-linking reagent or a condensation reagent.
 40. Ameasuring chip according to claim 6, wherein said immunoglobulin-bindingprotein is immobilized on said plasma polymerization layer through across-linking reagent or a condensation reagent.
 41. A measuring chipaccording to claim 8, wherein said sugar-binding protein is immobilizedon said plasma polymerization layer through a cross-linking reagent or acondensation reagent.
 42. A measuring chip according to claim 10,wherein said sugar-recognizing sugar chain is immobilized on said plasmapolymerization layer through a cross-linking reagent or a condensationreagent.
 43. A measuring chip according to claim 11, wherein said fattyacid or fatty acid ester is immobilized on said plasma polymerizationlayer through a cross-linking reagent or a condensation reagent.
 44. Ameasuring chip according to claim 13, wherein said polypeptide oroligopeptide is immobilized on said plasma polymerization layer througha cross-linking reagent or a condensation reagent.
 45. The measuringchip according to claim 38, wherein said cross-linking reagent is one ormore compounds selected from the group consisting of glutaraldehyde,periodic acid, N-succinimidyl-2-maleimidoacetic acid,N-succinimidyl-4-maleimidobutyric acid,N-succinimidyl-6-maleimidohexanic acid,N-succinimidyl-4-maleimidomethylcyclohexan-1-carboxylic acid,N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid,N-succinimidyl-4-maleimidomethylbanzoic acid,N-succinimidyl-3-maleimidobenzoic acid, N-sulfosuccinimidyl-3maleimidobenzoic acid, N-succinimidyl-4-maleimidophenyl-4-butyric acid,N-sulfosuccinimidyl-4-maleimidophenyl-4-butyric acid,N,N′-oxydimethylene-dimaleimide, N,N′-o-phenylene-dimaleimide,N,N′-m-phenylene-dimaleimide, N,N′-p-phenylene-dimaleimide,N,N′-hexamethylene-dimaleimide, N-succinimidylmaleimidocarboxylic acid,N-succinimidyl-S-acetylmercaptoacetic acid,N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinicanhydride, methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido,bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acidstilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon,1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,p-azidophenacylbromide, p-azidophenylglyoxal,N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate,N-(4-maleimidobutyloxy)sulfosuccinimide sodium salt,N-(6-maleimidocaproyloxy)sulfosuccinimide sodium salt,N-(8-maleimidocaproyloxy)sulfosuccinimide sodium salt, N-(11-maleimidoundecanoyloxy)sulfosuccinimide sodium salt,N-[2-(1-piperazinyl)ethyl] maleimide bichloric acid, bisdiazobenzidine,hexamethylenediisocyanate, toluenediisocyanate,hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimide,N,N′-polymethylenebisiodoacetamide, 2,4-dinitrobenzenesulfonate sodiumsalt, and diazo compounds; or said condensation reagent is one or morecompounds selected from the group consisting of carbodiimide derivativesrepresented by RN═C═NR (or R′), N-hydroxysuccinimide, tri-n-butylamine,butyl chloroformate, and isobutyl isocyanide.
 46. A measuring chipaccording to claim 1, wherein said immune protein or enzyme isimmobilized on said plasma polymerization layer through a cross-linkingreagent or a water-soluble condensation reagent.
 47. A measuring chipaccording to claim 46, wherein said cross-linking reagent is one or morecompounds selected from the group consisting of glutaraldehyde,N-succinimidyl-4-maleimidomethylbanzoic acid,N-succinimidyl-3-maleimidobenzoic acid,N-succinimidyl-4-maleimidophenyl-4-butyric acid,N,N′-oxydimethylene-dimaleimide, N,N′-m-phenylene-dimaleimide,N,N′-p-phenylene-dimaleimide, N,N′-hexamethylene-dimaleimide,N-succinimidylmaleimidocarboxylic acid,N-succinimidyl-S-acetylmercaptoacetic acid,N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinicanhydride, methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido,bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acidstilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon,1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,p-azidophenacylbromide, p-azidophenylglyoxal,N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate,bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate,hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimido,N,N′-polymethylenebisiodoacetoamide, and diazo compounds; or saidcondensation reagent is one or more compounds selected from the groupconsisting of carbodiimide derivatives represented by RN═C═NR (or R′),N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, andisobutyl isocyanide.
 48. The measuring chip for a surface plasmonresonance sensor of claim 1, further comprising a substance immobilizedon the surface of said plasma polymerization layer, and an additionalplasma polymerization layer or plasma-treated layer formed on saidplasma polymerization layer.
 49. A measuring chip according to claim 48,wherein said substance to be immobilized is selected from the groupconsisting of a non-immune protein, an immunoglobulin-binding protein, asugar-binding protein, a sugar-recognizing sugar chain, a fatty acid ora fatty acid ester, a polypeptide or oligopeptide having ligand-bindingactivity, an immune protein, and an enzyme.
 50. The measuring chip for asurface plasmon resonance sensor of claim 1, further comprising asubstance immobilized on said plasma polymerization layer through ahydrophobic bond.
 51. The measuring chip according to claim 50, whereinsaid substance to be immobilized is selected from the group consistingof a a non-immune protein, an immunoglobulin-binding protein, asugar-binding protein, a sugar-recognizing sugar chain, a fatty acid ora fatty acid ester, a polypeptide or oligopeptide having ligand-bindingactivity, an immune protein, and an enzyme.
 52. A method for producing ameasuring chip for a surface plasmon resonance sensor comprising:forming a metal layer on an optically transparent substrate, forming oneor more plasma polymerization layers on said metal layer, andimmobilizing a physiologically active substance on the surface of saidplasma polymerization layer.
 53. The method of claim 52, comprisingimmobilizing a non-immune protein on the surface of said plasmapolymerization layer.
 54. The method of claim 52 comprising immobilizingan immunoglobulin-binding protein on the surface of said plasmapolymerization layer.
 55. The method of claim 52 comprising Aimmobilizing a sugar-binding protein on the surface of said plasmapolymerization layer.
 56. The method of claim 52 comprising asugar-recognizing sugar chain on the surface of said plasmapolymerization layer.
 57. The method of claim 52 comprising _immobilizing a fatty acid or fatty acid ester on the surface of saidplasma polymerization layer.
 58. The method of claim 52 comprisingimmobilizing a polypeptide or oligopeptide having a ligand bindingcapability on the surface of said plasma polymerization layer.
 59. Amethod according to claim 52 wherein the plasma polymerization layer isformed by a plasma-treatment using a monomer material.
 60. The method ofclaim 52 comprising immobilizing an immune protein or enzyme on thesurface of said plasma polymerization layer.
 61. A measuring cell for asurface plasmon resonance sensor comprising a measuring chip accordingto claim
 1. 62. A measuring cell according to claim 61, wherein saidchip is optically analyzed.
 63. A surface plasmon resonance biosensorcomprising a measuring chip according to claim
 1. 64. A surface plasmonresonance biosensor comprising a measuring cell according to claim 61.